We will develop automated microfluidic devices for the high throughput measurements of kinase activation in human cells. These devices will ultimately be capable of detecting and characterizing the states of signal transduction pathways in cells by measuring and comparing the activity of multiple different kinases simultaneously in a large number of individual cells. The devices will automatically perform all of the processing and measurement steps required for high throughput measurements including cell transport, loading of kinase substrate peptides into the cells, cell lysis, and the separation and detection of the released peptides. The ratios of phosphorylated to nonphosphorylated substrates will be used as a measure of the degree of activation of each kinase assayed in each cell. Kinases play crucial roles in virtually all aspects of cellular physiology and are the focus of intense basic and pharmaceutical science research, thus these measurements have widespread applicability in the biomedical sciences. Moreover, by virtue of the heterogeneity of cellular behavior among individual cells in a population, high throughput single cell assays are critical for understanding how signaling networks interact to control cell physiology. Developing a technology, therefore, that is capable of determining the number, identities, and degree of activation of kinases in single cells will substantially advance our understanding of the molecular mechanisms of cellular signaling in health and disease.